Dual regulation and redundant function of two eye specific enhancers of the Drosophila retinal determination gene dachshund

2895

Introduction

The compound eye of Drosophila melanogasteris composed of a regular hexagonal array of 750 to 800 individual light-sensing ommatidia (Wolff and Ready, 1993). The initiation of eye development in Drosophila is controlled by a set of conserved tissue-specific genes. These genes, twin-of-eyeless (toy), eyeless (ey), eyes absent (eya), sine oculis (so) and dachshund (dac), function in a complex genetic regulatory hierarchy called the retinal determination (RD) network (Bonini et al., 1993; Cheyette et al., 1994; Czerny et al., 1999; Mardon et al., 1994; Quiring et al., 1994). The precise regulation of RD gene expression is crucially dependent on the integration of extracellular signaling inputs with cell-autonomous cues. Transcriptionally, such integration is largely directed by non-coding DNA sequences that include promoters and enhancers (Arnosti, 2003; Kadonaga, 2004). Enhancers are usually non-coding DNA stretches within the genome that are bound directly by upstream transcription factors and can regulate gene expression from a distance (Blackwood and Kadonaga, 1998). Transcription factors that bind enhancers are regulated by either an extracellular signal or are signal independent.

The adult Drosophila eye develops from an epithelial

monolayer called the eye imaginal disc, which is derived from a group of about 20 cells set aside during embryonic development (Garcia-Bellido and Merriam, 1969). Photoreceptor differentiation begins at the posterior margin of the eye disc in third instar larvae and proceeds anteriorly following a dorsoventral groove termed the morphogenetic furrow (MF) (Ready et al., 1976). The RD network consists of a series of gene regulatory events, which are initially linear and then progress to include extensive cross and feedback regulation, resulting in the conversion of undifferentiated epithelial cells to retinal cells (Chen et al., 1997; Halder et al., 1998; Pignoni et al., 1997). In addition to the cell-autonomously acting RD genes, extracellular signaling molecules such as Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) are also required for coordinating growth, proliferation, patterning and cell fate specification during retinal morphogenesis in Drosophila (Baonza and Freeman, 2002; Borod and Heberlein, 1998; Chanut and Heberlein, 1997; Dominguez and Hafen, 1997; Heberlein et al., 1995; Heberlein et al., 1993; Pignoni and Zipursky, 1997b; Treisman and Rubin, 1995).

dacis the most downstream member of the RD network to be identified in Drosophila (Chen et al., 1997). dac-null mutants in Drosophiladevelop with severely truncated legs and

Drosophila eye development is controlled by a conserved network of retinal determination (RD) genes. The RD genes encode nuclear proteins that form complexes and function in concert with extracellular signal-regulated transcription factors. Identification of the genomic regulatory elements that govern the eye-specific expression of the RD genes will allow us to better understand how spatial and temporal control of gene expression occurs during early eye development. We compared conserved non-coding sequences (CNCSs) between five Drosophilids along the ~40 kb genomic locus of the RD gene dachshund (dac). Our analysis uncovers two separate eye enhancers in intron eight and the 3′non-coding regions of the daclocus defined

by clusters of highly conserved sequences. Loss- and gain-of-function analyses suggest that the 3′ eye enhancer is synergistically activated by a combination of eya, so and

dppsignaling, and only indirectly activated by ey, whereas the 5′eye enhancer is primarily regulated by ey, acting in concert with eyaand so. Disrupting conserved So-binding sites in the 3′eye enhancer prevents reporter expression in vivo. Our results suggest that the two eye enhancers act redundantly and in concert with each other to integrate distinct upstream inputs and direct the eye-specific expression of dac.

Key words: dac, Enhancer, Eye, Drosophila, Retina

Summary

Dual regulation and redundant function of two eye-specific

enhancers of the Drosophila retinal determination gene dachshund

Mardelle R. Atkins1, Richard Gibbs2,4and Graeme Mardon1,2,3,5,6,*

1_{Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA}

2_{Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA} 3_{Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA}

4_{Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA} 5_{Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA} 6_{Department of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA}

*Author for correspondence (e-mail: gmardon@bcm.tmc.edu) Accepted 13 April 2005

Development 132, 2895-2905

Published by The Company of Biologists 2005 doi:10.1242/dev.01869

Research article

De

dramatically reduced or absent eyes (Mardon et al., 1994). In addition, dac mutants display defects in genital disc, mushroom body and antennal development (Dong et al., 2001; Dong et al., 2002; Kurusu et al., 2000; Martini et al., 2000; Noveen et al., 2000). Misexpression of dac is sufficient to induce ectopic eye development in non-retinal tissue (Shen and Mardon, 1997). dacencodes a nuclear protein that contains a conserved domain (Dachshund Domain 1 or DD1) which resembles DNA-binding motifs similar to those found in the winged helix/forkhead subfamily of helix turn helix proteins (Kim et al., 2002). In addition, a second conserved domain (Dachshund domain 2 or DD2) in Dac can form a complex with Eya, although recent studies have suggested that DD2 is largely dispensable for Dac protein function in vivo (Chen et al., 1997; Tavsanli et al., 2004). Dac is expressed in multiple tissues during Drosophila development, including the embryo, eye, leg, wing, antenna, male and female genital discs, and the mushroom bodies in the brain (Keisman and Baker, 2001; Kurusu et al., 2000; Mardon et al., 1994; Martini et al., 2000; Noveen et al., 2000). In the eye disc, Dac is expressed at the posterior margin prior to the initiation of the MF. After initiation of photoreceptor differentiation, Dac is expressed in the MF and its expression tapers both anterior and posterior to the furrow (Mardon et al., 1994).

Genetic analysis suggests that Dac expression in the eye is controlled by other members of the RD gene network. Dac expression is lost in eya or so mutant eye discs, and misexpression of ey or eya, but not so alone, leads to the inappropriate activation of Dac expression (Chen et al., 1997). Moreover, ectopic expression of a combination of eyaand so leads to the synergistic activation of Dac (Chen et al., 1999). Furthermore, dpp signaling can strongly synergize with eya and so to dramatically activate the expression of Dac in an ectopic expression assay, and dpp is required for dac expression in the eye disc (Chen et al., 1999). Last, the ability of ey to activate Dac expression is highly reduced but not completely eliminated in eya2 _{mutants (Chen et al., 1997).}

Taken together, these results suggest that dac regulation is under the control of ey, eyaand socoupled with extracellular inputs from Dpp signaling. Despite a host of genetic data, the exact nature of the protein complexes that regulate dac expression in the eye are still unknown. It has been proposed that So acts as the DNA binding unit of a protein complex that includes Eya, which in turn is thought to act as a transactivator (Chen et al., 1997). Furthermore, the roles of ey and downstream effectors of dppsignaling in the regulation of dac expression in the eye remain to be characterized.

The isolation of genomic elements that direct the eye-specific expression of the RD genes provide important tools for deciphering the molecular interactions that regulate early eye specification and determination. The eye enhancers of ey, eya, and so have been defined in some detail (Bui et al., 2000; Hauck et al., 1999; Niimi et al., 1999; Punzo et al., 2002; Zimmerman et al., 2000). These studies used eye-specific alleles of these genes to identify genomic lesions that disrupt regulatory elements that direct transcription in the eye. However, despite multiple attempts, no eye-specific alleles of dachave been isolated to date. Therefore, we turned to the use of functional genomics to identify the eye-specific regulatory elements of the dacgene in Drosophila. We hypothesized that crucial cis-regulatory non-coding sequences are highly

sensitive to mutational changes and remain largely unaltered over millions of years of evolution. Therefore, significant conservation in non-coding sequences among evolutionarily disparate species is a strong indicator of functional constraint and often uncovers cis-regulatory elements. We compared the sequences of the ~40 kb dac genomic region among five different species of Drosophilids to uncover highly conserved non-coding sequences (CNCSs). Two such CNCSs define eye-specific regulatory elements in the dac genomic locus. We demonstrate that one of these eye enhancers maps to the 3′ non-coding region of the daclocus and is under the genetic control of eya, so and dppsignaling. Two potential So-binding sites are embedded within an ~40 bp conserved stretch in this 3′eye enhancer and disruption of these binding sites abolishes enhancer activity in vivo. Surprisingly, in spite of the 3′eye enhancer being completely deleted in dac7_{homozygotes, these}

animals develop with only moderately disrupted eyes. Our genomic analysis identifies a second, independent 5′ eye enhancer that maps to intron 8 of the daclocus and that acts redundantly and in concert with the 3′eye enhancer. This 5′ eye enhancer is not deleted in dac7mutants and is regulated by a combination of ey, eyaand so. Our results highlight the power of functional genomics to uncover genomic regulatory elements, especially in the absence of tissue-specific genetic mutants and in cases with redundant enhancers.

Materials and methods

Comparative genomics

Drosophila erecta and willistoni clones were isolated from the BACPAC Resources 50 kb fosmid libraries. High-density filters were probed with a labeled fragment of exon 2 of D. melanogaster dac using standard hybridization techniques. For D. virilis, this probe was used on an amplified lambda library provided by Ron Blackman and Thomas Kaufman (Thummel, 1993). Positive clones were fingerprinted and end sequenced. Appropriate clones were shotgun sequenced to ~10 coverage. Conserved regions were identified using the BLASTZ program using the command line parameters H=2200 K=2200 to increase sensitivity (Schwartz et al., 2003). D. pseudoobscura sequence was obtained by BLAST searches at http://www.hgsc.bcm.tmc.edu/projects/drosophila/. A BioPerl script was used to mask coding regions, to find conserved non-coding sequences (CNCSs) in all clones, to cluster nearby CNCSs together and to perform T-COFFEE alignments of all CNCS blocks (Notredame et al., 2000; Stajich et al., 2002). Graphic representations of enhancer conservation were generated using the AVID/mVISTA server at http://www.gsd.lbl.gov/vista/ (Bray et al., 2003; Dubchak et al., 2000; Frazer et al., 2004; Mayor et al., 2000).

Drosophila genetics

All Drosophilacrosses were carried out at 25°C on standard media. The mad1-2 FRT40A recombinant stock was provided by Marek Mlodzik (Curtiss and Mlodzik, 2000). The nature of the dac3and dac7 mutant alleles were previously described (Tavsanli et al., 2004). The presence of intron 8 in dac7 _{mutants was confirmed by PCR on}

genomic DNA prepared from dac7_{homozygotes with intron 8 specific}

primers. A similar assay was used to demonstrate the deletion of exon 9, placing the deletion in dac7_{beyond intron 8 but including exon 9}

(data not shown). The 30A-GAL4, UAS-ey,UAS-eyaand UAS-soflies were previously described (Brand and Perrimon, 1993; Pignoni et al., 1997). UAS-eya and UAS-so stocks were provided by Francesca Pignoni and Larry Zipursky. All other stocks were obtained from the Bloomington stock center. Flies containing multiple transgenes were generated by meiotic recombination using eye color as an initial

De

selection. Polymerase chain reaction (PCR) with gene-specific primers was used to confirm genotypes. Ectopic expression followed by antibody staining (where possible) was used to confirm expression of individual genes from recombinant chromosomes.

P-element vectors and reporter transgene construction

Genomic fragments spanning the dac locus were subcloned into appropriate P-element reporter vectors using convenient restriction sites. Three different P-element reporter vectors were used in this study: pCasper-hs43-AUG-βGal(Thummel et al., 1988), pH-Pelican and pH-Stinger(Barolo et al., 2000). The reporters in pH-Pelicanand pH-Stinger are β-galactosidase and nuclear GFP, respectively. To generate an HA-dac version of the enhancer-reporter construct, we deleted the entire GFP-coding region from the pH-Stinger vector and replaced it with an HA tag in frame with the daccDNA. This vector still contains the 390 bp eye enhancer and a minimal hsp70 TATA promoter. Detailed information about this vector is available upon request.

Sub-fragments of 1 kb or less were obtained by PCR amplification using appropriate primers with artificial EcoRI-BamHI restriction site tails. PCR products were digested with EcoRI and BamHI, and ligated with similarly digested P-element vectors. Positive clones were sequenced to confirm sequence integrity and orientation. Fragments with mutated binding sites were obtained by overlap extension PCR as previously described (Ho et al., 1989). Subcloned PCR products were sequenced to confirm the sequence and orientation. Transgenic flies were obtained by standard transgenic injection techniques (Rubin and Spradling, 1982). A minimum of three independent transgenic lines were tested for reporter activity for each construct.

β-Galactosidase activity staining

Imaginal discs from second or third instar larvae were dissected into phosphate buffered saline [PBS; 0.1 M phosphate (pH 7.2), 150 mM NaCl], fixed for 20 minutes in 1% glutaraldehyde in PBS, and washed three times for 10 minutes each in PBS. The imaginal discs were then incubated in pre-warmed active staining solution (10 mM Na2HPO4, 10 mM NaH2PO4, 150 mM NaCl, 1 mM MgCl2, 3 mM K3[Fe(CN)6], 3 mM K4[Fe(CN)6]) with 0.1% X-gal in N,N-dimethylformamide. The discs were allowed to stain for appropriate times up to 16 hours and then washed in PBS three times for 10 minutes each wash. The discs were allowed to equilibrate in 80% glycerol in PBS overnight before they were mounted on glass slides.

Immunohistochemistry and scanning electron microscopy

Primary antibodies used in this study were: monoclonal mouse anti-Dachshund (mAbdac2-3: 1:200, Developmental Studies Hybridoma Bank), rabbit anti-β-galactosidase (1:1000; Cappel), rabbit anti-GFP (Molecular Probes), chicken anti-GFP (Upstate) and mouse anti-HA (Covance). Conjugated goat anti-mouse, chicken and rabbit fluorescent secondary antibodies were ALEXA 488 (Molecular Probes), Cy3 (Jackson Immunochemicals) or Cy5 (Jackson Immunochemicals), all at 1:600 dilution. HRP-conjugated goat anti-mouse antibodies were used as previously described (Mardon et al., 1994). Discs were then processed as previously described (Frankfort et al., 2001). Fluorescent images were captured with a Zeiss LSM 510 confocal microscope. All other images were captured on a Zeiss Axioplan microscope with Nomarski optics. All images were processed with Adobe Photoshop software. Adult flies were prepared for electron microscopy as previously described (Kimmel et al., 1990).

Results

The 3′non-coding region of the dac locus contains an eye-specific enhancer

To understand the molecular regulation of dac in various

tissues, we sought to uncover the genomic regulatory elements that control dacexpression. The genomic organization of the daclocus is shown in Fig. 1A. daccomprises 12 exons and the last exon is separated from the 3′ neighboring Idgf gene complex by ~13.3 kb of non-coding genomic DNA. The 5′ neighbor, predicted gene CG4580, is separated by 2.2 kb from the first exon of dac. The 5′gene tpr2is 3.7 kb upstream of the first coding exon of dac (not shown). Prior to using a functional genomics approach to uncover novel enhancer elements in the daclocus, we generated transgenic flies that carry large genomic fragments spanning the entire daclocus cloned upstream of a minimal, heat shock protein(hsp) TATA promoter driving a β-galactosidase reporter (Thummel et al., 1988). Third instar imaginal discs from these transgenic lines were then tested for β-galactosidase activity. We found that a 16.6 kb NotI-SpeI, genomic fragment from the 3′end of the daclocus contained reporter activity in patterns reminiscent of endogenous dactranscript and protein expression in the eye, lamina, leg, antenna and wing (Fig. 1A and data not shown; see Materials and methods). Using restriction sub-fragments that span this 16.6 kb region, we were able to narrow the eye-lamina enhancer to a 1.9 kb fragment that contains eye-specific reporter activity posterior to the morphogenetic furrow (3EE1.9 kb_{; see Fig. S1A in the supplementary material). All the}

3′ eye enhancer fragments are henceforth denoted by 3EE followed by their length in superscript. Six overlapping, PCR generated sub-fragments that span the 1.9 kb eye enhancer were then tested for reporter activity. A 390 bp sub-fragment (3EE390 bp) within the 1.9 kb fragment contains eye enhancer activity. Further dissection of the 3EE390 bp _{fragment with}

smaller PCR fragments uncovered a 194 bp eye reporter fragment (3EE194 bp_{; see Fig. S1A in the supplementary}

material). However, all the eye-specific enhancer fragments described above drive reporter expression only posterior to the MF in the eye, suggesting that these fragments lack important sequences that regulate dacexpression anterior to the MF (Fig. 1D and data not shown).

We then used a functional genomics approach to uncover new genomic non-coding sequences across the entire the dac locus that are required for tissue specific enhancer activity (see Materials and methods). We hypothesized that non-coding regions that remain unaltered over the course of millions of years of evolutionary time are under functional constraint and define important regulatory protein binding targets. We compared the conservation of non-coding DNA across the ~40 kb dac genomic locus among five related species of Drosophilids, D. melanogaster, D. pseudoobscura, D. erecta, D. willistoni, and D. virilisthat represent over 60 million years of evolutionary time (see Materials and methods). As we were primarily interested in uncovering eye enhancer fragments, we initially focused on sequences within 3EE1.9 kb. The VISTA output of pairwise comparisons to D. melanogaster along 3EE1.9 kb is shown in Fig. 1C (Mayor et al., 2000). Six conserved non-coding sequences (CNCSs) are present in 3EE1.9 kb. To test the correlation of CNCSs with enhancer activity, we cloned an 850 bp fragment (3EE850 bp) that contains all six CNCS blocks upstream of a minimal promoter driving expression of a GFP or β-galactosidase reporter. Transgenic flies were then tested for reporter (GFP or β-galactosidase) expression in the eye. 3EE850 bp, like 3EE1.9 kb, is expressed only posterior to the furrow (Fig. 1D). However, a smaller 659

De

bp fragment (3EE659 bp_{) that contains only the first four CNCS}

blocks drives strong expression of GFP in the eye disc both anterior and posterior to the furrow, similar to endogenous Dac protein expression (Fig. 1E). The smallest active enhancer

3EE194 bp contains only the third and fourth CNCS blocks, and is expressed only posterior to the MF in third instar eye discs (data not shown). These results suggest that 3EE659 bp (expressed both anterior and posterior to the MF) lacks repressor binding sites contained in the 1.9 kb eye enhancer that normally inactivate reporter expression anterior to the furrow. In addition, these results suggest that 3EE194 bp_{(expressed only}

posterior to the MF) further lacks positive regulatory sites normally present in 3EE659 bpthat are required for reporter expression anterior to the MF. Although the expression of endogenous Dac protein is decreased posterior to the furrow, GFP expression driven by 3EE659 bp_persists

all the way to the posterior margin of the eye disc. To rule out the possibility that the 3′ enhancer lacks repressive elements that normally downregulate dacexpression posterior to the furrow, we generated transgenic flies in which the GFP reporter was replaced by an HA-dac reporter (see Materials and methods). HA-Dac reporter expression, visualized using an anti-HA antibody, reveals a rapid downregulation of HA staining posterior to the MF (see Fig. S1B-D in the supplementary material). Thus, we conclude that GFP expression far posterior to the MF in 3EE-GFP transgenic eye discs occurs because of the perdurance of GFP protein and/or transcript, and not because of the lack of negative regulatory elements in the 3′ enhancer. We hypothesized that a deletion of the 3′eye enhancer would block dac expression in the eye, thereby causing eye-specific defects. We therefore examined known dac mutants to identify an allele that contains genomic lesions in this 3′eye enhancer but does not affect the function of the protein.

The dac7_{mutant contains a} large deletion in the 3′region of the dac genomic locus that includes the 3′enhancer

The eye enhancers of ey, eya and so have been defined through eye-specific alleles of these genes (Cheyette et al., 1994; Quiring et al., 1994; Zimmerman et al., 2000). Such eye-specific mutants often disrupt genomic regulatory sequences that direct expression of the transcript to the eye imaginal disc. Despite two large-scale F1 genetic

Fig. 1.Conserved non-coding sequences in the daclocus uncover two eye enhancers. (A) The dachshundgenomic locus, with 5′and 3′eye enhancers indicated (see text for details). (B) AVID/mVISTA representation of the 5′eye enhancer (5EE). The numbered peaks indicate four areas of significant conservation. A small inversion in D. willistonimasks the first two peaks. (C) AVID/mVISTA representation of the 3′eye enhancer (3EE). The numbered peaks indicate six areas of significant conservation and two sub-fragments tested are shown. 3EE850 bp contains all six CNCS blocks and is expressed only posterior to the MF. 3EE659 bpcontains the first four CNCS blocks and is expressed both anterior and posterior to the MF. The smallest active enhancer fragment identified is 194 bp (3EE194 bp) and contains CNCS blocks 3 and 4. Regions of significant conservation are indicated in pink (B,C), and the predicted Sine oculis-binding site is highlighted in bright blue and is within CNCS block 3. (D,E) Representative third instar eye discs from 3EE850 bp-GFP (D) and 3EE659 bp-GFP (E) larvae triple labeled with GFP (green, D,E), Sens (magenta, D′,E′) and Dac (blue, D′′,E′′). GFP expression in 3EE659 bpeye discs is detected anterior to the earliest Sens expression and overlaps with anterior Dac

expression (E′,E′′). By contrast, GFP expression in 3EE850 bpeye discs is not detected anterior to the anterior-most Sens-expressing column (D′).

De

screens over deficiencies spanning the dac locus, we have been unable to isolate eye-specific alleles of dac. However, in a previous study aimed at analyzing the structure and function of the conserved domains of the Dac protein, we molecularly characterized several dac mutant alleles to identify coding region mutants that truncate the Dac protein prematurely (Tavsanli et al., 2004). One such allele, dac7, is a large deletion in the daclocus that begins in exon 9 and extends beyond the neighboring Idgf genes (Fig. 2A; the distal extent of this deletion has not been mapped). In light of our finding that the 3′non-coding region of dac contains cis-regulatory elements, dac7 mutants provide us with a tool to analyze the role of this 3′eye enhancer in an in vivo context. We hypothesized that dac7 mutants should be severe hypomorphs or null mutants owing to the lack of cis-regulatory elements. Furthermore, as the first eight exons are intact in dac7mutants, we predicted that any dac7 transcript would encode a protein with an intact N terminus, DD1 and middle region. Previous structure-function analyses suggest that such a truncated protein is functional in vivo and can completely rescue dac3null mutants (Tavsanli et al., 2004).

dac7homozygotes develop with only moderately disrupted eyes

Surprisingly, dac7homozygotes develop with only moderately disrupted eyes compared with wild-type adults (compare Fig. 2C with 2B). By contrast, dac3 null mutants have no eyes, suggesting that the dac7 mutant is a hypomorph (Fig. 2D). We also examined the expression of Dac protein in the eye imaginal discs of dac7 homozygous larvae. A monoclonal antibody to Dac (mabdac 2-3) recognizes an epitope predicted to be present within the potentially truncated protein encoded by the dac7 transcript. Eye imaginal discs from dac7 larvae are almost identical to wild-type controls in their Dac protein expression profiles (compare Fig. 2F to 2E). dac3 -null mutants display no detectable Dac protein (Fig. 2G). As the entire 16.6 kb 3′ enhancer is completely deleted in dac7 mutants, these results suggest that additional eye-specific enhancers exist in the genome, either within the dac locus or outside the genomic fragments we tested.

A second eye enhancer is present in intron 8 of the dac genomic locus

We next extended our pairwise sequence comparison to the entire dac genomic locus to identify additional functionally relevant CNCSs. Multiple regions of significant conservation were found, spread along the entire locus (data not shown). We used PCR amplification to clone these CNCS-containing fragments upstream of a β-galactosidase reporter. One such fragment contains four CNCS blocks in a 1.7 kb stretch within intron 8 of the daclocus (called 5′eye enhancer or 5EE; Fig. 1B). Importantly, this 1.7 kb region is intact in the dac7allele.

We found that third instar eye discs from 5EEtransgenic larvae are positive for β-galactosidase activity, which appears to be highest at the posterior margin of the eye disc (Fig. 2M). Furthermore, late first instar and second instar 5EEtransgenic eye discs also have β-galactosidase activity, suggesting that this enhancer is active prior to initiation of the MF (Fig. 2L; data not shown). A smaller fragment that contains only the first two CNCS blocks does not have eye enhancer activity (data not shown). Taken together, these results suggest that another eye enhancer exists in intron 8 of the daclocus that perhaps acts redundantly or in concert with the 3′enhancer. We next tested

Fig. 2.The 3′eye enhancer is dispensable for dacactivation in vivo. (A) dac7_is

associated with a deletion, beginning at exon 9, that uncovers the entire 3′ genomic region of dac. (B-D) Scanning electron micrographs (SEMs) of adult eyes from wild-type (B), dac7_{(C) and dac}3_{(D) animals. (E-G) Third instar eye}

imaginal discs from wild type (E), dac7_{(F) and dac}3_{(G) animals stained with a}

monoclonal Dac antibody, mab2-3. dac7_{eye discs show relatively normal Dac}

protein expression when compared with wild-type eye discs (compare F with E). dac7_{adult eyes are rough and disorganized but still contain ~50% of the normal}

number of ommatidia (compare C with B). By comparison, dac3_homozygotes

express no Dac protein (G) and develop with no eyes (D). (H,I) A first instar (H) and second instar (I) eye disc of wild type shown for comparison with J and L. (J-M) Late first (J) and third (K) instar eye imaginal discs from 5EE-lacZand second (L) and third (M) instar eye imaginal discs from 3EE-lacZtransgenic larvae. β -Galactosidase activity is detected in both first instar and third instar 5EE-lacZeye discs, primarily at the posterior margin (J,K). The earliest 3EE-lacZreporter expression is observed in second instar eye discs and this β-galactosidase activity persists in the third instar eye disc (L,M).

De

the response of these putative eye enhancers to known upstream regulators of dacin the Drosophilaeye.

The 3′dac eye-specific enhancer is regulated by dpp, eya and so

Many studies have shown that dac expression in the eye is regulated by upstream members of the RD network such as ey, eya and so (Chen et al., 1997; Chen et al., 1999; Shen and Mardon, 1997). We tested whether any of these upstream factors could activate the expression of either the 3′enhancer or the 5′(intron 8) enhancer in an ectopic expression assay. All 3′eye enhancer fragments tested respond identically in these ectopic assays and are described 3EEfor simplicity (data are shown only for the 3EE659 bpfragment). We used the previously described 30A-Gal4line in this ectopic expression assay as it drives the expression of UAS-transgenes in a ring around the wing pouch (Chen et al., 1999; Pappu et al., 2003). so alone does not activate reporter expression or endogenous Dac in this assay (Chen et al., 1999) (data not shown). However, either ey oreyaexpressed alone can activate endogenous Dac and 3EE-GFPin this assay, but only in regions of the wing that express dpp endogenously (Fig. 3A; data shown only for ey misexpression). Expression of a combination of eya and so induces synergistic expression of 3EE-GFP, but this induction is also limited to regions that coincide with endogenous dpp expression (Fig. 3B). Thus, the 3′ eye enhancer is activated similarly to endogenous dacin this ectopic expression assay.

Previous studies have shown that dpp signaling acts synergistically with eya and so, and strongly activate dac expression in the 30A-Gal4 ectopic expression assay (Chen et al., 1999). We tested if 3EE-GFP is also synergistically activated by a combination of eya, soand dppin the ectopic wing expression assay. As with endogenous Dac protein, the expression of 3EE-GFPwas strongly induced in a ring around the wing pouch upon expression of dpp, eyaand so using the 30-Gal4driver (Fig. 3C). These results suggest that the 3′dac eye-specific enhancer may be directly regulated by a combination of Dpp signaling effector molecules and upstream RD proteins. Furthermore, these results suggest that 3EE194 bp is sufficient to integrate the input from Dpp signaling with the tissue-specific factors Eya and So. Interestingly, the intracellular transducers of Dpp signaling, Mothers against Dpp (Mad) and Medea, do not bypass the requirement for Dpp in this assay (data not shown). However, a constitutively active form of the Dpp receptor, Thickveins (TkvQ253D), was just as effective as Dpp in synergistically activating GFP expression from the 3′eye enhancer in the presence of Ey (Lecuit et al., 1996) (data not shown). Therefore, we conclude that the ability of Dpp to synergize with Eya and So to activate 3EE is dependent on downstream signaling events such as the phosphorylation of Mad. A less probable alternative is that non-canonical events downstream of Tkv mediate the synergy between Eya, So and Dpp signaling. We found no evidence for autoregulation of 3EEby Dac itself, as ectopic expression of Dac with the 30-Gal4 driver does not activate reporter expression in the wing (data not shown).

ey acts through eya and so to regulate the 3′dac eye enhancer

Results from our ectopic expression analysis suggested that the 3′daceye enhancer is regulated by a combination of eyaand

so. Consistent with this prediction, 3EE-GFP expression is completely lost in eya2 _{and so}1 _{eye-specific mutants (Fig.}

4B,C). However, as has been shown previously, endogenous Dac protein expression is dramatically reduced but not completely eliminated in eya2and so1mutants (Fig. 4A-C). As our ectopic expression data suggest that dppsignaling acts in concert with eya and so to activate 3EE, we tested the expression of 3EE-GFPin eye imaginal discs cells that have lost the ability to signal downstream of the dppreceptor tkv. To disrupt dpp signaling, we induced mad mutant mitotic clones in the eye disc using a strong hypomorphic allele of mad (mad1-2_{). We found that 3EE-GFP expression is drastically}

reduced or completely lost from posterior margin mad1-2clones (Fig. 4D). These loss- and gain-of-function experiments suggest that 3EEis regulated by a combination ofeya, soand dpp. Coupled with the ectopic expression data, we conclude that 3EEactivation is dependent on the canonical dppsignaling pathway acting synergistically with eyaand so.

eycan also activate endogenous Dac protein and 3EE-GFP in an ectopic expression assay using the 30A-Gal4driver. As ectopic eyexpression activates eyaand soexpression in regions where dpp signaling is present (Chen et al., 1999) and ey directly activates soexpression (Niimi et al., 1999; Punzo et

Fig. 3.Synergistic activation of 3EE-GFP by eya, soand dpp. (A-C) Each set of three panels shows the same wing disc stained with anti-Dac (magenta) and anti-GFP (green) or a merge of the two channels. (A) Wing imaginal discs in which the expression of UAS-eyis driven by 30A-Gal4show ectopic expression of 3EE-GFP and Dac in two regions at the anteroposterior (AP) compartment boundary. (B) Wing imaginal discs expressing a combination of eya and sodriven by 30A-Gal4can strongly induce 3EE-GFP and Dac at the AP compartment boundary where their expression coincides with endogenous dpp(white). (C) A combination of dpp, eyaand so driven by 30A-Gal4synergistically induces the expression of 3EE -GFP and Dac in the entire ring around the wing pouch.

De

al., 2002), we predicted that 3EE-GFPis indirectly activated by eyin ectopic expression assays via the induction of eyaand so. We tested this hypothesis by determining if ectopic ey expression could activate 3EE-GFP in eya2 and so1 mutant backgrounds in an ectopic expression assay. We used the dpp-Gal4driver in this assay, which drives UAS-eyexpression in

all imaginal discs including the ventral antennal disc (Shen and Mardon, 1997). dpp-Gal4 driven ey expression can induce endogenous Dac protein expression and 3EE-GFPexpression in the ventral antenna (Fig. 4E,F). However, in eya2or so1

Fig. 4.eya, soand dppsignaling are required for regulation of 3EE-GFP. (A-C) Each set of three panels shows the same eye disc stained with anti-Dac (magenta in A-C) and anti-GFP (green in A′-C′) or a merge of the two channels (A′′-C′′). (A-A′′) Wild-type eye imaginal discs stained with GFP and Dac reveal the normal expression of endogenous Dac (A) and 3EE-GFP (A′). (B-C′) so1_{(B,B′) and eya}2

(C,C′) eye imaginal discs have drastically reduced levels of Dac (B,C) and completely lack 3EE-GFP (B′,C′). (D-D′′′) Each set of four panels shows the same eye disc stained with anti-β -galactosidase (magenta in D), anti-Dac (green in D′), anti-GFP (green in D′′), or a merge of the three channels (D′′′). Posterior margin madmutant clones, negatively marked by the lack of β -galactosidase, block Dac (D′) and GFP (D′′) expression. (E,F) An eye-antennal disc from a w; UAS-ey, 3EE-GFP; dpp-Gal4third instar larva stained with an antibody against GFP alone (E) or GFP and Dac (F). Ectopic eyexpression in the antenna driven by dpp-Gal4 can strongly induce 3EE-GFP (E) and Dac (F) in the ventral antenna (arrows). (G,H) Eye-antennal discs from w; eya2_{; dpp-Gal4,}

3EE-GFP/UAS-ey(G) and w; so1_{; dpp-Gal4, 3EE-GFP/UAS-ey(H) larvae}

co-stained with antibodies against GFP and Dac (both panels show a merge of the two channels). Ectopic eyexpression in the antenna (arrows) driven by dpp-Gal4cannot induce 3EE-GFP expression (green in G and H) but retains the ability to induce Dac expression in the ventral antenna (magenta in G and H).

Fig. 5.5EE-lacZis regulated by ey, eyaand so. (A-J) All panels show wing (A-F) or eye-antennal (G-J) discs from 5EE-lacZ transgenic lines stained to reveal β-galactosidase reporter activity. (A) A wing imaginal disc from a 5EE-lacZthird instar larva shows weak enhancer activity (present in multiple transgenic lines). (B,C) Wing imaginal discs from 5EE-lacZthird instar larvae in which the expression of UAS-eyalone (B) or a combination of UAS-dpp, UAS-eyaand UAS-so(C) is driven by 30A-Gal4. (B) eyalone, but not (C) a combination of UAS-dpp, UAS-eyaand UAS-so, is capable of inducing 5EE-lacZin the ring around the wing disc. (D-F) Wing imaginal discs from 5EE-lacZthird instar larvae in which the expression of UAS-eyis driven by dpp-Gal4in wild-type (D), so1_{(E), eya}2_{(F) or mutant backgrounds. Ectopic eyexpression}

is able to induce β-galactosidase reporter expression via 5EEat the AP boundary in all three cases. The activity is stronger in so1_mutant

wing discs than eya2_{mutant wing discs. (G,H) Eye-antennal}

imaginal discs from so1_{(G) or eya}2_{(H) mutant 5EE-lacZthird instar}

larvae stained for β-galactosidase activity. No reporter activity is detected in these mutant eye discs. (I,J) Eye-antennal imaginal discs from w; so1_{; dpp-Gal4, 5EE-lacZ/UAS-eyand w; eya}2_{; dpp-Gal4,}

5EE-lacZ/UAS-eythird instar larvae. Strong induction of 5EE-lacZis seen in the ventral antenna in both so1_{and eya}2_{mutants (arrowheads}

in I and J, respectively). In addition, β-galactosidase activity is restored at the posterior margin of so1_{mutant eye discs (arrow in I).}

De

mutant antennal discs,dpp-Gal4 driven ectopic eyexpression can induce endogenous Dac, but not 3EE-GFPexpression (Fig. 4G,H). These results suggest that ey regulation of 3EE is achieved indirectly by the activation of eyaand so.

The 5′dac eye enhancer is activated by ey, eya and so

We next tested if the putative 5′ eye enhancer 5EE could respond to ectopic expression of upstream regulators of dac expression such as ey, eya, soand dpp. We used both the 30A-Gal4and dpp-Gal4drivers in ectopic expression assays to test the response of 5EE to upstream factors in the wing disc. Surprisingly, although ectopic expression of eydriven by the 30A-Gal4 driver induces 5EE-lacZ in the posterior compartment of the wing disc, a combination of dpp, eyaand so does not induce expression of 5EE-lacZ in the 30-Gal4 ectopic expression assay (Fig. 5B,C). Similarly, ectopic expression of eydriven by the dpp-Gal4 driver induces 5EE-lacZexpression at the anteroposterior compartment boundary of the wing disc (Fig. 5D). We then tested whether eyactivation of 5EEis also indirect via the activation of eyaand so. Unlike 3EE-GFP, which is not induced by eyin eya2or so1mutants, ectopic ey expression is able to induce 5EE-lacZ reporter expression even in eya2or so1mutant wing discs (Fig. 5E,F). These results suggest that ey acts directly, or through other factors independent of eya and so, to regulate 5EE-lacZ. However, we noticed that ectopic ey-mediated induction of 5EE-lacZis not as robust in eya2mutant wing discs as in wild-type wing discs, suggesting that the activity of ey is compromised in the absence of eyafunction.

Our ectopic expression analyses suggest that eyis the primary upstream regulator of 5EE-lacZ. Furthermore, these results suggest that eyaand soare dispensable for 5EEactivity in vivo. Surprisingly, we found that 5EEexpression is lost in third instar so1 _{and eya}2 _{eye imaginal discs (Fig. 5G,H). These results}

suggest that although eya and so are not sufficient to regulate

5EE, they are required for eyto regulate this enhancer. However, as ectopic eycan activate 5EEin eya2and so1 mutant wing discs, we hypothesized that high levels of ey are sufficient to circumvent the requirement for eyaand so. To test this hypothesis directly, we made use of the unusual nature of the dpp-Gal4driver to activate eyexpression in the eye. Although normal dpp expression mirrors movement of the MF, expression of dpp-Gal4is limited to the posterior margin of the eye imaginal disc (Shen and Mardon, 1997). We used the dpp-Gal4driver to drive eyexpression ineyaor somutant eye discs. Consistent with ectopic eyexpression in the wing, dpp-Gal4 driven expression of ey restores 5EE-lacZexpression at the posterior margin of so1 mutant eye discs (Fig. 5I). However, dpp-Gal4 driven expression of ey can induce only weak expression of 5EE-lacZin eya2mutant eye discs (Fig. 5J). These results suggest that the activity of the 5′eye enhancer is primarily regulated by ey. Furthermore, these results suggest that the function of eyin this context is more sensitive to the levels of eyathan so.

Two conserved So binding sites are essential for normal expression of the 3′eye enhancer

The smallest fragment in the 3′ dac eye enhancer that can respond to dpp, eya and so is 3EE194 bp, which is centered around two CNCS blocks of ~40 bp and 20 bp (Fig. 1C). These two CNCS blocks are also common to all active fragments of the 3′ eye enhancer. We scanned these two evolutionarily conserved stretches for known, genetically upstream transcription factor binding sites. We found that the 40 bp conserved stretch contains two putative consensus So-binding sites, S1-5′-CGATAT and S2-5′-CGATAC, compared with the consensus 5′-(C/T)GATA(C/T) described previously (Hazbun et al., 1997; Yan et al., 2003) (Fig. 6A). We mutated each of these putative So-binding sites in 3EE individually and in combination to test their requirement for normal enhancer activity in vivo (Fig. 6A). Mutation of individual So-binding sites causes a severe reduction, but not complete elimination, of enhancer activity in vivo (Fig. 6C,D). However, simultaneous mutation of both So binding sites completely abolishes enhancer activity in vivo (Fig. 6E). These results, coupled with loss-and gain-of-function analyses with dpp, eya and so, suggest that So binds to the 3′eye enhancer directly and nucleates a protein complex that includes Eya to regulate 3EE. However, despite much effort using a wide variety of binding conditions, we have been unable to demonstrate specific, direct binding of So protein to oligos that contain these So-binding sites. We also scanned the 5′eye enhancer, which has four CNCS blocks, for potential upstream transcription factor binding sites and found no strong candidate binding sites within the CNCS blocks.

Discussion

The precise spatial and temporal specificity of gene expression

Fig. 6.Mutating the putative So binding sites in 3EEabolishes enhancer activity in vivo. (A) A multiple sequence alignment of the 40 conserved bases in the 3′eye enhancer. Mismatched bases are shown in grey and the two putative So-binding sites are shown in green. Mutated So-binding sites are shown in red. (B-E) Each panel shows a single eye disc co-stained with anti-Dac (blue) and anti-GFP (green). Mutating each putative So-binding site individually (C,D) results in the dramatic reduction of GFP reporter expression from the 3EEenhancer when compared with the wild-type version of same enhancer (B). When both So-binding sites are mutated, enhancer activity and GFP expression is completely abolished (E). Endogenous Dac expression is unaffected in all cases.

De

is often governed by coupling tissue-specific inputs with signaling from extracellular growth and patterning factors such as Dpp, Hh and Wg. In particular, it has been proposed that the sequential induction and repression of a small subset of genes allows the formation of specialized protein complexes that in turn activate progressively refined gene expression programs. dac is the most downstream member of the RD network identified so far. Isolation of the genomic regulatory elements of dacprovide an opportunity to study the interplay between intracellular transcription factors and downstream effectors of signaling pathways to control RD gene expression. In this study, we use functional genomics to uncover two independent genomic regulatory sequences that direct the expression of dac to the eye. Both eye enhancers are located within non-coding genomic regions that exhibit significant sequence conservation among five species of Drosophilids separated by ~65 million years of evolutionary time. Our results suggest that significant conservation in non-coding genomic regions is a strong predictor of regulatory function. In the absence of easily available genetic reagents, in silico functional genomics approaches provide efficient tools to uncover the complexity of gene regulation across phylogeny.

Dual regulation of dac expression: the roles of the 5′ and 3′eye enhancers

Loss- and gain-of-function analyses with the two eye enhancers suggest that each enhancer is regulated by a distinct set of protein complexes. The 5′eye enhancer is activated by a combination of ey, eya and so, but is not activated by Dpp signaling. 5EEis activated by ectopic eyexpression even in eya and so mutants, suggesting that it is regulated exclusively by ey. However, somewhat paradoxically, 5EEexpression is lost in eya and so mutants even though ectopic expression of a combination of dpp, eyaand sodoes not activate this enhancer. Furthermore, driving high levels of eyin so1mutant eye discs restores 5EE-lacZ expression. Coupled together, these results suggest that 5EE is primarily regulated by ey but that the regulation of 5EEby eyalso requires eyaand so.

By contrast, the 3′ dac eye enhancer is regulated by a combination of eya, so and dppsignaling, but is not directly dependent on ey. 3EE-GFPexpression is lost in eya2and so1 mutant eye discs, and in posterior margin mad1-2_{mutant clones.}

Furthermore, eycannot bypass the requirement for eyaand so to activate 3EE. Conversely, 3EEis strongly induced by co-expression of eyaand so. Moreover, dppsignaling via the tkv receptor can synergize with eyaand soto induce 3EEin ectopic expression assays. Furthermore, we find that neither Mad nor Medea, the intracellular transducers of Dpp signaling, is sufficient to bypass the requirement for activation of the Dpp receptor Tkv in these assays (data not shown). Thus, we conclude that events downstream of Dpp-Tkv signaling, such as the phosphorylation of Mad, are essential for the synergistic activation of the 3′ dac eye enhancer by eya and so. Taken together, these results suggest that there are distinct requirements for the activation of the 5′ and 3′ dac eye enhancers. However, the exact nature of the protein complexes that regulate 5EEand 3EEremain to be determined.

Initiation versus maintenance of dac expression: the roles of the 5′and 3′eye enhancers

MF initiation is completely blocked in posterior margin dac3

-null mutant clones. However, dac3clones that do not include any part of the posterior margin develop do not prevent MF progression, but cause defects in ommatidial cell number and organization (Mardon et al., 1994). This dichotomy in dac function is reflected in the two eye enhancers we have characterized in this study. Our analysis of dac7_homozygotes

demonstrates that the 3′ eye enhancer is dispensable for MF initiation and progression. We propose that in dac7 _mutants,

the intact 5EEenhancer is sufficiently activated by eyto drive high enough levels of dacexpression to initiate and complete retinal morphogenesis. However, dac7 mutants have readily observable defects in ommatidial organization. Thus, we further propose that this lack of normal patterning in dac7 mutants is most likely due to the loss of 3EE, which normally acts in concert with 5EE after MF initiation, to integrate patterning inputs from extracellular signaling molecules such as Dpp with tissue-specific upstream regulators such as ey, eya and so. However, we do not know if the 3′ eye enhancer is sufficient to initiate dacexpression in the absence of the 5′eye enhancer.

Based on our results, we propose a two-step model for the regulation of dacexpression in the eye. First, the initiation of dacexpression in the eye disc is dependent on Ey binding to 5EE. However, Ey is fully functional only when So and Eya are present. It is possible that Ey recruits So and Eya to 5EE, but we favor a model in which Ey bound to 5EE cooperates with an So/Eya complex bound to 3EE to initiate dac expression in the eye. After initiation of the MF, dacexpression is maintained by an Eya and So complex bound to 3EE. In addition, 3EEcan integrate patterning information received via dpp signaling, thereby allowing the precise spatial and temporal expression of dacin the eye. This two part retinal enhancer ensures that dacexpression is initiated only after ey activates eya and so expression. Thus, the daceye enhancers provide a unique model with which the sequential activation of RD proteins allows the progressive formation of specialized protein complexes that can activate retinal specific genes.

The redundancy in dacenhancer activity also explains our inability to isolate eye-specific alleles of dac, despite multiple genetic screens (K.S.P., E.J.O. and G.M., unpublished). The modular nature of the two enhancers and their potential ability to act independently or in concert suggest that both enhancers must be disrupted to block high levels of transcription of dac. Thus, two independent hits in the same generation, a phenomenon that occurs infrequently in genetic screens, would be required to obtain an eye-specific allele in dac.

The dac eye enhancers provide powerful tools with which to study RD protein function

Despite much investigation, very few direct targets of RD proteins, especially for Eya and So, have been identified. One study suggests that So can bind to and regulate an eye-specific enhancer of the lzgene (Yan et al., 2003). However, lzis not expressed early during eye development and is required only for differentiation of individual cell types (Daga et al., 1996). Our results suggest that regulation of dacexpression occurs via the interaction of two independent eye enhancers that are likely to be bound by Ey, Eya and So, and respond to dppsignaling. Our analysis of the 3′eye enhancer suggests that two putative conserved So-binding sites are essential for 3EE activity in vivo. Mutation of individual So-binding sites dramatically

De

reduces, but does not completely eliminate, reporter expression in the eye. Mutating both predicted So-binding sites completely blocks enhancer activity in vivo. Thus, we conclude that So binds to 3EE via these conserved binding sites. However, we have not been able to demonstrate a direct specific interaction of either So alone or a combination of Eya and So with oligos that contain these putative So-binding sites in vitro. It is possible that other unidentified proteins are required for stabilizing the Eya and So complex. Furthermore, the 194 bp fragment that responds to ectopic expression of dpp, eya, and so contains no conserved or predicted Mad-binding sites. This raises the intriguing possibility that dppsignaling activates other genes, which then directly act with eyaand so to regulate the 3′eye enhancer. Alternatively, a large complex that includes Eya, So and the intracellular transducers of dpp signaling, such as Mad and Medea, may be responsible for activation of 3EE. Similarly, our results suggest that the 5′eye enhancer is regulated primarily by ey. However, it is unclear whether Ey directly binds 5EE. Furthermore, Ey is fully functional only in the presence of Eya and So. Thus, Ey either independently recruits Eya and So into a 5′ complex or is activated by virtue of its proximity to the So/Eya complex bound to the 3′enhancer or both.

The exact order and dynamics of protein complex assembly at 5EE and 3EE requires further investigation. However, the two daceye enhancers are extremely useful tools with which to investigate fundamental issues about the mechanism of RD protein action. One significant issue concerns the mechanism of Eya function during eye development. Eya consists of two major conserved domains, an N-terminal domain that has phosphatase activity in vitro and a C-terminal domain that can function as a transactivator in cell culture assays (Rayapureddi et al., 2003; Silver et al., 2003; Tootle et al., 2003). So contains a conserved Six domain and a DNA binding homeodomain (Cheyette et al., 1994; Kawakami et al., 2000). However, it is unclear if Eya provides phosphatase activity, transactivator function, or both, in this complex. Characterization of the components of the protein complexes that regulates dac expression may uncover the targets of Eya phosphatase activity during eye development. Thus, the isolation of two eye enhancers with distinct regulation provides very useful tools with which to study protein complex formation and function during Drosophilaretinal specification and determination.

We thank Marek Mlodzik, Gary Struhl, Francesca Pignoni, Nancy Bonini, Larry Zipursky, Hugo Bellen, the Bloomington stock center and the Developmental Studies Hybridoma Bank for providing fly stocks and antibodies. We thank Benjamin Frankfort and Richard Davis for discussions and comments on the manuscript. Research in the laboratory of G.M. is supported by funds from the National Eye Institute (R01 EY11232), the Retina Research Foundation and an Ophthalmology Core Grant from the National Eye Institute (EY02520). M.R.A. is supported by NEI training grant T32 EY07102.

Supplementary material

Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/12/2895/DC1

References

Arnosti, D. N.(2003). Analysis and function of transcriptional regulatory elements: insights from Drosophila. Annu. Rev. Entomol.48, 579-602. Baonza, A. and Freeman, M.(2002). Control of Drosophilaeye specification

by Wingless signalling. Development129, 5313-5322.

Barolo, S., Carver, L. A. and Posakony, J. W. (2000). GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila. Biotechniques29, 726-732.

Blackwood, E. M. and Kadonaga, J. T.(1998). Going the distance: a current view of enhancer action. Science281, 61-63.

Bonini, N. M., Leiserson, W. M. and Benzer, S.(1993). The eyes absent gene: genetic control of cell survival and differentiation in the developing Drosophila eye. Cell72, 379-395.

Borod, E. R. and Heberlein, U.(1998). Mutual regulation of decapentaplegic

and hedgehog during the initiation of differentiation in the Drosophila

retina. Dev. Biol.197, 187-197.

Brand, A. H. and Perrimon, N.(1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development118, 401-415.

Bray, N., Dubchak, I. and Pachter, L.(2003). AVID: a global alignment program.Genome Res.13, 97-102.

Bui, Q. T., Zimmerman, J. E., Liu, H., Gray-Board, G. L. and Bonini, N. M.(2000). Functional analysis of an eye enhancer of the Drosophila eyes absentgene: differential regulation by eye specification genes. Dev. Biol.

221, 355-364.

Chanut, F. and Heberlein, U.(1997). Role of decapentaplegicin initiation and progression of the morphogenetic furrow in the developing Drosophila

retina. Development124, 559-567.

Chen, R., Amoui, M., Zhang, Z. and Mardon, G.(1997). Dachshund and eyes absent proteins form a complex and function synergistically to induce ectopic eye development in Drosophila. Cell91, 893-903.

Chen, R., Halder, G., Zhang, Z. and Mardon, G.(1999). Signaling by the TGF-beta homolog decapentaplegic functions reiteratively within the network of genes controlling retinal cell fate determination in Drosophila.

Development126, 935-943.

Cheyette, B. N., Green, P. J., Martin, K., Garren, H., Hartenstein, V. and Zipursky, S. L. (1994). The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron12, 977-996.

Curtiss, J. and Mlodzik, M.(2000). Morphogenetic furrow initiation and progression during eye development in Drosophila: the roles of

decapentaplegic, hedgehogand eyes absent. Development127, 1325-1336. Czerny, T., Halder, G., Kloter, U., Souabni, A., Gehring, W. J. and Busslinger, M.(1999). twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development. Mol. Cell.3, 297-307.

Daga, A., Karlovich, C. A., Dumstrei, K. and Banerjee, U. (1996). Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with mAML1. Genes Dev.10, 1194-1205.

Dominguez, M. and Hafen, E.(1997). Hedgehog directly controls initiation and propagation of retinal differentiation in the Drosophilaeye. Genes Dev.

11, 3254-3264.

Dong, P. D., Chu, J. and Panganiban, G. (2001). Proximodistal domain specification and interactions in developing Drosophila appendages.

Development128, 2365-2372.

Dong, P. D., Dicks, J. S. and Panganiban, G. (2002). Distal-less and

homothoraxregulate multiple targets to pattern the Drosophilaantenna.

Development129, 1967-1974.

Dubchak, I., Brudno, M., Loots, G. G., Pachter, L., Mayor, C., Rubin, E. M. and Frazer, K. A.(2000). Active conservation of noncoding sequences revealed by three-way species comparisons.Genome Res.10, 1304-1306. Frankfort, B. J., Nolo, R., Zhang, Z., Bellen, H. and Mardon, G.(2001).

senseless repression of rough is required for R8 photoreceptor differentiation in the developing Drosophila eye. Neuron32, 403-414. Frazer, K. A., Pachter, L., Poliakov, A., Rubin, E. M. and Dubchak, I.

(2004). VISTA: computational tools for comparative genomics. Nucleic Acids Res.32, W273-W279.

Garcia-Bellido, A. and Merriam, J. R.(1969). Cell lineage of the imaginal discs in Drosophilagynandromorphs. J. Exp. Zool.170, 61-75.

Halder, G., Callaerts, P., Flister, S., Walldorf, U., Kloter, U. and Gehring, W. J.(1998). Eyeless initiates the expression of both sine oculis and eyes absent during Drosophila compound eye development. Development125, 2181-2191.

Hauck, B., Gehring, W. J. and Walldorf, U.(1999). Functional analysis of an eye specific enhancer of the eyelessgene in Drosophila. Proc. Natl. Acad. Sci. USA 96, 564-569.

Hazbun, T. R., Stahura, F. L. and Mossing, M. C.(1997). Site-specific

De

recognition by an isolated DNA-binding domain of the sine oculis protein.

Biochemistry36, 3680-3686.

Heberlein, U., Wolff, T. and Rubin, G. M.(1993). The TGF beta homolog

dppand the segment polarity gene hedgehogare required for propagation of a morphogenetic wave in the Drosophilaretina. Cell75, 913-926. Heberlein, U., Singh, C. M., Luk, A. Y. and Donohoe, T. J.(1995). Growth

and differentiation in the Drosophilaeye coordinated by hedgehog. Nature

373, 709-711.

Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R.(1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene77, 51-59.

Kadonaga, J. T.(2004). Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors. Cell116, 247-257.

Kawakami, K., Sato, S., Ozaki, H. and Ikeda, K. (2000). Six family genes–structure and function as transcription factors and their roles in development. BioEssays22, 616-626.

Keisman, E. L. and Baker, B. S.(2001). The Drosophilasex determination hierarchy modulates wingless and decapentaplegic signaling to deploy

dachshundsex-specifically in the genital imaginal disc. Development128, 1643-1656.

Kim, S. S., Zhang, R. G., Braunstein, S. E., Joachimiak, A., Cvekl, A. and Hegde, R. S. (2002). Structure of the retinal determination protein Dachshund reveals a DNA binding motif. Structure 10, 787-795. Kimmel, B. E., Heberlein, U. and Rubin, G. M.(1990). The homeo domain

protein rough is expressed in a subset of cells in the developing Drosophila

eye where it can specify photoreceptor cell subtype. Genes Dev.4, 712-727. Kurusu, M., Nagao, T., Walldorf, U., Flister, S., Gehring, W. J. and Furukubo-Tokunaga, K.(2000). Genetic control of development of the mushroom bodies, the associative learning centers in the Drosophilabrain, by the eyeless, twin of eyeless, and dachshundgenes. Proc. Natl. Acad. Sci. USA 97, 2140-2144.

Lecuit, T., Brook, W. J., Ng, M., Calleja, M., Sun, H. and Cohen, S. M. (1996). Two distinct mechanisms for long-range patterning by

Decapentaplegicin the Drosophila wing. Nature381, 387-393.

Mardon, G., Solomon, N. M. and Rubin, G. M.(1994). dachshundencodes a nuclear protein required for normal eye and leg development in

Drosophila. Development120, 3473-3486.

Martini, S. R., Roman, G., Meuser, S., Mardon, G. and Davis, R. L.(2000). The retinal determination gene, dachshund, is required for mushroom body cell differentiation. Development127, 2663-2672.

Mayor, C., Brudno, M., Schwartz, J. R., Poliakov, A., Rubin, E. M., Frazer, K. A., Pachter, L. S. and Dubchak, I.(2000). VISTA: visualizing global DNA sequence alignments of arbitrary length. Bioinformatics 16, 1046-1047.

Niimi, T., Seimiya, M., Kloter, U., Flister, S. and Gehring, W. J.(1999). Direct regulatory interaction of the eyelessprotein with an eye-specific enhancer in the sine oculis gene during eye induction in Drosophila.

Development126, 2253-2260.

Notredame, C., Higgins, D. G. and Heringa, J.(2000). T-Coffee: a novel method for fast and accurate multiple sequence alignment.J. Mol. Biol.302, 205-217.

Noveen, A., Daniel, A. and Hartenstein, V.(2000). Early development of the

Drosophila mushroom body: the roles of eyeless and dachshund.

Development127, 3475-3488.

Pappu, K. S., Chen, R., Middlebrooks, B. W., Woo, C., Heberlein, U. and Mardon, G.(2003). Mechanism of hedgehogsignaling during Drosophila

eye development. Development130, 3053-3062.

Pignoni, F. and Zipursky, S. L. (1997b). Induction of Drosophila eye development by decapentaplegic. Development124, 271-278.

Pignoni, F., Hu, B., Zavitz, K. H., Xiao, J., Garrity, P. A. and Zipursky, S. L.(1997). The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophilaeye development. Cell91, 881-891. Punzo, C., Seimiya, M., Flister, S., Gehring, W. J. and Plaza, S.(2002).

Differential interactions of eyelessandtwin of eyelesswith the sine oculis

enhancer. Development129, 625-634.

Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J.(1994). Homology of the eyeless gene of Drosophilato the Small eye gene in mice and Aniridia in humans. Science265, 785-789.

Rayapureddi, J. P., Kattamuri, C., Steinmetz, B. D., Frankfort, B. J., Ostrin, E. J., Mardon, G. and Hegde, R. S.(2003). Eyes Absent represents a class of protein tyrosine phosphatases. Nature426, 295-298.

Ready, D. F., Hanson, T. E. and Benzer, S.(1976). Development of the Drosophila retina, a neurocrystalline lattice. Dev. Biol.53, 217-240.

Rubin, G. M. and Spradling, A. C. (1982). Genetic transformation of

Drosophilawith transposable element vectors. Science218, 348-353. Schwartz, S., Kent, W. J., Smit, A., Zhang, Z., Baertsch, R., Hardison, R.

C., Haussler, D. and Miller, W.(2003). Human-mouse alignments with BLASTZ.Genome Res.13, 103-107.

Shen, W. and Mardon, G.(1997). Ectopic eye development in Drosophila

induced by directed dachshundexpression. Development124, 45-52. Silver, S. J., Davies, E. L., Doyon, L. and Rebay, I. (2003). Functional

dissection of eyes absent reveals new modes of regulation within the retinal determination gene network. Mol. Cell. Biol. 23, 5989-5999.

Stajich, J. E., Block, D., Boulez, K., Brenner, S. E., Chervitz, S. A., Dagdigian, C., Fuellen, G., Gilbert, J. G., Korf, I., Lapp, H. et al.(2002). The Bioperl toolkit: Perl modules for the life sciences.Genome Res.12, 1611-1618.

Tavsanli, B. C., Ostrin, E. J., Burgess, H. K., Middlebrooks, B. W., Pham, T. A. and Mardon, G.(2004). Structure-function analysis of the Drosophila

retinal determination protein Dachshund. Dev. Biol. 272, 231-247. Thummel, C. (1993). Compilation of Drosophila cDNA and genomic

libraries. Dros. Info. Serv. 72, 180-183.

Thummel, C. S., Boulet, A. M. and Lipshitz, H. D.(1988). Vectors for

Drosophila P-element-mediated transformation and tissue culture transfection. Gene74, 445–456.

Tootle, T. L., Silver, S. J., Davies, E. L., Newman, V., Latek, L. R., Mills, I. A., Selengut, J. D., Parlikar, B. E. W. and Rebay, I. (2003). The transcription factor Eyes Absentis is a protein tyrosine phosphatases. Nature

426, 299-302.

Treisman, J. E. and Rubin, G. M.(1995). winglessinhibits morphogenetic furrow movement in the Drosophilaeye disc. Development121, 3519-3527. Wolff, T. and Ready, D. F.(1993). Pattern formation in the Drosophilaretina. In The development of Drosophila melanogaster, vol. II (ed. M. Bate and A. Martinez-Arias), pp. 1277-1325. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Yan, H., Canon, J. and Banerjee, U.(2003). A transcriptional chain linking eye specification to terminal determination of cone cells in the Drosophila eye. Dev. Biol. 263, 323-329.

Zimmerman, J. E., Bui, Q. T., Liu, H. and Bonini, N. M.(2000). Molecular genetic analysis of Drosophila eyes absentmutants reveals an eye enhancer element. Genetics154, 237-246.

De